The utility of transgenic tools that can selectively label each particular type of neuron, and furthermore, selectively inactivate genes or manipulate the activity of one type of neuron at a time, has high impact in many different areas of neuroscience research. Current approaches taken by many, e.g. the Cre-driver line creation, usually utilize genes relatively specifically expressed in certain cell types. However, such "marker" genes rarely label a really specific neuronal population. It is highly desirable to further refine the target specificity into a particular region or a specific population of neurons. To achieve this we propose to systematically evaluate a number of combinatorial strategies and develop a series of transgenic tools to drive highly specific gene expression through the intersection of the expression of 2 marker genes. Through mining our genome-wide database, Allen Brain Atlas, Dr. Zeng and colleagues have identified a large set of gene markers for different cortical cell types. Dr. Nagy has developed a set of gene targeting and allele replacement strategies for expressing multiple genes efficiently. Allen Institute has a high throughput ISH platform and capability of high resolution image acquisition and databasing. By combining the superb technologies developed by both parties, we will examine the colocalization of different marker genes by double fluorescent ISH (dFISH), create both driver and reporter/responder mouse lines to test different intersection strategies, and use the dFISH again to systematically characterize and database where the controlled gene expression occurs in the entire mouse brain. In this grant we will use the neocortex as a model to establish the technology. Once completed, it can be applied easily into other brain regions as well. PUBLIC HEALTH RELEVANCE: Studying the extreme complexity of the brain demands ways to tease apart its components. Traditional transgenic tools have been widely used in every area of neuroscience research. Our proposed research will provide two types of powerful tools to the neuroscience community: transgenic mouse lines that can direct genetic manipulation to highly specific neuronal populations, and an image database comprehensively documenting where such manipulation occurs. Both of these tools will have major impact on the broad neuroscience community, from molecular to systems neuroscience, from development to behavior studies, from studies of normal brain physiology to disease mechanisms.